Hitherto, it has been well known that in a method for purification and separation of a nucleic acid from a nucleic acid-containing material, a nucleic acid mixture is adsorbed on particles of glass and silica gel, glass, silica wool, silica, a glass membrane, a polymer or the like under the presence of chaotropic agents. The nucleic acid-containing material is mainly a biological raw material such as cultured cells and tissues, body fluids including blood, serum, urine and feces, germs including bacteria and human Mycobacterium tuberculosis, or viruses of HIV, hepatitis B and hepatitis C, and plasmid DNA, genomic DNA, chromosomal DNA, RNA, mitochondrial DNA, fragment DNA and the like can be separated and purified.
Purification of fragment DNA is a technique that is very frequently used in molecular biological studies, and conducts prior to PCR, cloning, sequencing, restriction enzyme digestion and applications of other enzymic actions and the like.
For example, there is a method of isolating DNA from a recombinant M13 phage, and an isolation method in which M13 phage DNA binds on a glass fiber filter by adding chaotropic agents and then eluted via separation, washing and drying is shown in Nucleic Acids Research Vol. 15 5507-5516 (1978).
In a method in which a glass powder is added, DNA binds to the glass powder, centrifugal separation is performed, the glass powder is collected and washed, and DNA is eluted and isolated is shown in Pvoc. Natl. Acad. Sci. USA Vol. 76, 615-619 (1979). A similar method is described in Japanese Patent Laid-Open No. 59-227744, Analytical Biochemistry Vol. 121.382-387 (1982), Molecular cloning: A Laboratory Manual 188-190 (1982) and so on.
A method in which a composite biological starting material, chaotropic agents and a nucleic acid binding solid phase including silica or a derivative thereof are mixed, the solid phase to which a nucleic acid binds is separated from a liquid, and washed to elute the nucleic acid is proposed in Japanese Patent No. 2680462.
The physical mechanism of adsorbing DNA and RNA under the presence of chaotropic agents has not been clarified for its details, but it is believed that a cation exchange reaction occurs between a negatively charged carrier and a nucleic acid. Thus, the efficiency of purification can be considered to be equal to the efficiency of contact between the surface of the carrier and a biological sample.
Irrespective of which of the above described carriers is used, a procedure in which a carrier on which a nucleic acid is to be adsorbed is held in a container (cartridge, chip or the like), and a biological sample is passed through the container, a nucleic acid is adsorbed on the carrier with an adsorption buffer solution, contaminants other than nucleic acid components are then expelled to outside the cartridge with a washing liquid, and an elution liquid is then passed to take out the nucleic acid components with the liquid is general.
In addition, purification of fragment DNA from an agarose gel by electrophoresis and various extractions is often performed, but this method requires much time, and the obtained DNA is extremely thin, contains salts and organic solvents, and is therefore required to be further desalted or concentrated by ethanol precipitation. In a conventional method such as a gel filtration purification technology, it is very difficult to separate molecules having similar molecular weights.
These separation methods using a carrier require use of high-concentration salts, and therefore a phenomenon causing decomposition or degradation of DNA on the surfaces of particles of glass or silica gel has been confirmed. The adsorption occurs substantially quantitatively under the presence of high-concentration chaotropic agents, but elution of adsorbed DNA is carried out under the presence of a buffer solution of the salts. The treatment of fractions of DNA is limited to a range of 100 base pairs (bp) to 10000 base pairs (bp), and it is impossible to quantitatively separate or purify DNA fractions with 100 base pairs (bp) or less or DNA with 10000 to 100000 base pairs (bp).
For a method based on a glass powder (glass beads) as a carrier, it is conceivable that beads are downsized or the amount of beads is increased for improving the efficiency of contact between the surface of the carrier and a nucleic acid. However, a pressure during passage of a liquid increases, the operability is considerably impaired, and air gaps between beads become so small that nucleic acid molecules cannot enter the air gaps because of their large molecular weight, resulting in a problem of reducing the efficiency contrarily. If the length of the container is increased for the purpose of improving the separation, the pressure increases, and in addition, the amount of eluted solvent increases to reduce the efficiency of concentration, leading to a process which is less convenient. Further, if the bead or wool is used, its fragments and particles enter an elution liquid although the amount is very small, thus causing a problem in a subsequent application. If a packing method for packing the carrier in a container is not fixed, separation time and the pattern are changed, and therefore a problem of poor stability of separation arises.
A method using a membrane or a filter as a carrier has an advantage that it can be processed for improved usability, but it is difficult to form pores appropriate to separation with control, and the method is thus poor in practicality.
A method using a polymer resin is not capable of purification with a simple protocol, since a separation system is complicated such that an action of reacting specifically with a nucleic acid depending on the property of the polymer resin or a part having an influence exists in addition to the concerned part.
In any case, high-purity fragment DNA cannot be purified. There are disadvantages of difficulty of handling unique to a powder silica resin and a suspension, hindrance of a subsequent application reaction, and so on.
As in the invention of Japanese Patent No. 2680462, the concept of direct isolation of a nucleic acid from a complicated starting material without a pretreatment refers to “digestion” and “purification” being performed at the same time. As a result, drawbacks of requiring a radical reaction condition and reducing a range of applicable molecular weights of nucleic acids are yielded.
In Japanese Patent laid-open No. Heisei 8-501321, a method is proposed in which a nucleic acid mixture is adsorbed on a porous or nonporous inorganic base such as silica gel or glass from an adsorption aqueous solution containing high-concentration (ionic strength) salts and an organic acid such as an aliphatic alcohol or polyethylene glycol, washed and then eluted with a solution containing lower-concentration (ionic strength) salts to obtain a nucleic acid.
However, in this method, a nucleic acid mixture is adsorbed on an inorganic material from an adsorption aqueous solution containing high-concentration salts and a nucleic acid is eluted with a solution containing salts although its concentration is low, and therefore if, for example, a DNA sample is included in large agarose fractions, it is necessary to treat the sample using a plurality of columns, and eluted fragment DNA is pooled, and further, obtained fragment DNA contains salts and organic solvents, and therefore operation steps of concentration, desalination and the like are required, and moreover, purified DNA may be lost during ethanol precipitation.